Mucin antigen vaccine

ABSTRACT

Provided are expression vectors for generating an immune response to a mucin. The vectors comprise a transcription unit encoding a secretable polypeptide, the polypeptide comprising a secretory signal, a mucin antigen and CD40 ligand. Also provided are methods of generating an immune response against cells expressing a mucin by administering an effective amount of the vector. Further provided are methods of generating an immune response against cancer cells expressing a mucin in an individual by administering an effective amount of the vector. Still further provided are methods of overcoming anergy to a mucin self antigen by administering an effective amount of the vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/662,616, filed on Oct. 29, 2012, which is a continuation of U.S.patent application Ser. No. 10/997,055, filed on Nov. 23, 2004, whichclaims priority to U.S. Provisional Patent Application No. 60/524,925,filed on Nov. 24, 2003, U.S. Provisional Patent Application No.60/525,552, filed on Nov. 25, 2003, and U.S. Provisional PatentApplication No. 60/529,015, filed on Dec. 11, 2003, from each of whichpriority is claimed, and the disclosures of which are all herebyincorporated herein by reference in their entireties, including alltables, figures, and claims.

GOVERNMENTAL RIGHTS

This invention was made with Government support under Contract NumberDAM017-99-1-9457 funded by the U.S. Army Medical Research and MaterialCommand's funding agreement to the Sidney Kimmel Cancer Center. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the development of immunity against amucin using a vector that expresses a secretable polypeptide comprisinga mucin antigen fused to CD40 ligand.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.This application claims priority to U.S. application Ser. Nos.60/524,925 (filed Nov. 24, 2003), 60/525,552 (filed Nov. 25, 2003), and60/529,015 (filed Dec. 11, 2003), all of which are incorporated hereinby reference in their entirety including the drawings. An applicationrelated to this application is PCT/US03/36237 filed Nov. 12, 2003entitled “adenoviral vector vaccine,” hereby incorporated by referencein its entirety including the drawings.

The activation of antigen presenting cells (APCs) which includes thedendritic cells (DCs), followed by loading of the antigen presentingcell with relevant antigens, is a requisite step in the generation of aT cell dependent immune response against cancer cells. Once activatedand loaded with tumor antigens, DCs migrate to regional lymph nodes(LNs) to present antigens to T cells. Very commonly, these APCs expressinsufficient amounts of surface activation molecules which are requiredfor optimal activation and expansion of T cell clones competent torecognize tumor antigens. See Shortman, et al., Stem Cells 15:409-419,1997.

Antigen presentation to naive T cells, in the absence of costimulatorymolecule expression on the surface of the APC, leads to anergy of the Tcells. See Steinbrink, et al. Blood 99: 2468-2476, 2002. Moreover,cross-presentation by DCs without CD4⁺ T cell help also results inperipheral deletion of Ag-specific T cells in regional LNs. SeeKusuhara, et al., Eur J Immunol 32:1035-1043, 2002. In contrast, in thepresence of CD4⁺ T cell help, DCs acquire functional ability tocross-prime T cells, resulting in clonal expansion of effector T cells.See Gunzer, et al., Semin Immunol 13:291-302, 2001. This CD4⁺ T cellhelp can be replaced with CD40-CD40 ligand (CD40L) interactions. SeeLuft, et al. Int Immunol 14:367-380, 2002. CD40L is a 33-kDa type IImembrane protein and a member of the TNF gene family and is transientlyexpressed on CD4⁺ T cells after TCR engagement. See Skov, et al. J.Immunol. 164: 3500-3505, 2000.

The ability of DCs to generate anti-tumor immune responses in vivo hasbeen documented in a number of animal tumor models. See Paglia, et al. JExp Med 183: 317-322, 1996; Zitvogel, et al., J Exp Med. 183: 87-97,1996. However, DC-mediated induction of immunity represents a majortherapeutic challenge. It is considered difficult to ensure that theantigen presenting cells express appropriate adhesion molecules andchemokine receptors to attract DCs to secondary lymphoid organs forpriming T cells. See Fong, et al. J. Immunol. 166: 4254-4259, 2001;Markowicz, et al. J Clin Invest. 85: 955-961, 1990; Hsu, et al. Nat.Med. 2: 52-58, 1996; Nestle, et al. Nat. Med. 4: 328-332, 1998; Murphy,et al., Prostate 38: 73-78, 1999; Dhodapkar, et al. J Clin Invest. 104:173-180, 1999.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an expression vector forgenerating immunity against a mucin. The vector includes a transcriptionunit encoding a secretable polypeptide that contains a secretory signalsequence, a mucin antigen and CD40 ligand. In a preferred embodiment,the CD40 ligand is human CD40 ligand.

In one approach, the sequence encoding the mucin antigen in thetranscription unit is 5′ to sequence encoding the CD40 ligand. Inanother approach, the sequence encoding the CD40 ligand in thetranscription unit is 5′ to sequence encoding the mucin antigen. In apreferred embodiment, the CD40 ligand lacks all or a portion of itstransmembrane domain.

In preferred embodiments, the expression vector may be a viralexpression vector or a non-viral expression vector; e.g., an adenoviralvector; the mucin antigen is from a mucin selected from the groupconsisting of MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7,MUC8, MUC9, MUC12, MUC13, MUC15, and MUC16; the mucin antigen is fromMUC1; the mucin antigen includes the extracellular domain of a mucin; orat least one tandem repeat of a mucin; and the transcription unitincludes sequence that encodes a linker between the tumor antigen andthe CD40 ligand. Suitable linkers may vary in length and composition.

In other embodiments, the expression vector includes a humancytomegalovirus promoter/enhancer for controlling transcription of thetranscription unit.

In another aspect, the invention provides methods for generating animmune response in an individual against cells expressing a mucinantigen by administering an effective amount of a vector that includes atranscription unit encoding a polypeptide containing, starting from theamino terminus, a secretory signal sequence, the mucin antigen and asecretable form of CD40 ligand.

In preferred embodiments, the cells are cancer cells; and the methodresults in the generation of cytotoxic CD8⁺ T cells against the mucin.

In yet another aspect, the invention provides methods for treating anindividual with cancer that expresses a mucin antigen. The methodincludes administering to the individual an effective amount of a vectorthat has a transcription unit encoding a mucin antigen and CD40 ligandcontaining polypeptide as described above.

In preferred embodiments, the cancer cells are carcinoma cancer cells.

In a further aspect, the invention provides a method for generating animmune response to a mucin in a human where the mucin is a human selfantigen and the immune cells of the individual are anergic to the mucin.The method includes administering to the individual an effective amountof a vector that has a transcription unit encoding a mucin antigen andCD40 ligand containing polypeptide as described above.

In the above methods, the vector is advantageously administeredsubcutaneously and may be given one or more subsequent times to increasethe immune response. The immunity against the antigen is long lastingand involves generation of cytotoxic CD8⁺ T cells.

Abbreviations used herein include “Ad” (adenoviral); “sig” (signalsequence); and “ecd” (extracellular domain).

These and other embodiments are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the nucleotide sequence encoding human MUC1 (SEQ IDNO:1)

FIG. 2 shows the amino acid sequence of human MUC1 (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, a vector is provided forgenerating immunity against a mucin antigen. The vector includes atranscription unit encoding a secretable polypeptide containing asecretory signal sequence, a mucin antigen and CD40 ligand. In apreferred embodiment, the transcription unit includes from the aminoterminus, a secretory signal sequence, a mucin antigen and a secretableform of CD40 ligand. In preferred embodiments, the secretable form ofCD40 ligand lacks all or substantially all of its transmembrane domain.

The term “vector” which contains a transcription unit (aka. “expressionvector”) as used herein refers to viral and non-viral expression vectorsthat when administered in vivo can enter target cells and express anencoded protein. Viral vectors suitable for delivery in vivo andexpression of an exogenous protein are well known and include adenoviralvectors, adeno-associated viral vectors, retroviral vectors, herpessimplex viral vectors, and the like. Viral vectors are preferably madereplication defective in normal cells. See U.S. Pat. Nos. 6,669,942;6,566,128; 6,794,188; 6,110,744; 6,133,029.

The term “adenoviral expression vector” as used herein, refers to anyvector from an adenovirus that includes exogenous DNA inserted into itsgenome which encodes a polypeptide. The vector must be capable ofreplicating and being packaged when any deficient essential genes areprovided in trans. An adenoviral vector desirably contains at least aportion of each terminal repeat required to support the replication ofthe viral DNA, preferably at least about 90% of the full ITR sequence,and the DNA required to encapsidate the genome into a viral capsid. Manysuitable adenoviral vectors have been described in the art. See U.S.Pat. Nos. 6,440,944 and 6,040,174 (replication defective E1 deletedvectors and specialized packaging cell lines). A preferred adenoviralexpression vector is one that is replication defective in normal cells.

Adeno-associated viruses represent a class of small, single-stranded DNAviruses that can insert their genetic material at a specific site onchromosome 19. The preparation and use of adeno-associated viral vectorsfor gene delivery is described in U.S. Pat. No. 5,658,785.

Non-viral vectors for gene delivery comprise various types of expressionvectors, (e.g., plasmids) which are combined with lipids, proteins andother molecules (or combinations of thereof) in order to protect the DNAof the vector during delivery. Fusigenic non-viral particles can beconstructed by combining viral fusion proteins with expression vectorsas described. Kaneda, Curr Drug Targets (2003) 4(8):599-602.Reconstituted HVJ (hemagglutinating virus of Japan; Sendaivirus)-liposomes can be used to deliver expression vectors or thevectors may be incorporated directly into inactivated HVJ particleswithout liposomes. See Kaneda, Curr Drug Targets (2003) 4(8):599-602.DMRIE/DOPE lipid mixture are useful a vehicle for non-viral expressionvectors. See U.S. Pat. No. 6,147,055. Polycation-DNA complexes also maybe used as a non-viral gene delivery vehicle. See Thomas et al., ApplMicrobiol Biotechnol (2003) 62(1):27-34.

The term “transcription unit” as it is used herein in connection with anexpression vector means a stretch of DNA that is transcribed as asingle, continuous mRNA strand by RNA polymerase, and includes thesignals for initiation and termination of transcription. For example, inone embodiment, a transcription unit of the invention includes nucleicacid that encodes from 5′ to 3,′ a secretory signal sequence, a mucinantigen and CD40 ligand. The transcription unit is in operable linkagewith transcriptional and/or translational expression control elementssuch as a promoter and optionally any upstream or downstream enhancerelement(s). A useful promoter/enhancer is the cytomegalovirus (CMV)immediate-early promoter/enhancer. See U.S. Pat. Nos. 5,849,522 and6,218,140.

The term “secretory signal sequence” (aka. “signal sequence,” “signalpeptide,” leader sequence,” or leader peptide”) as used herein refers toa short peptide sequence, generally hydrophobic in charter, includingabout 20 to 30 amino acids which is synthesized at the N-terminus of apolypeptide and directs the polypeptide to the endoplasmic reticulum.The secretory signal sequence is generally cleaved upon translocation ofthe polypeptide into the endoplasmic reticulum. Eukaryotic secretorysignal sequences are preferred for directing secretion of the exogenousgene product of the expression vector. A variety of suitable suchsequences are well known in the art and include the secretory signalsequence of human growth hormone, immunoglobulin kappa chain, and thelike. In some embodiments the endogenous mucin signal sequence also maybe used to direct secretion.

The term “tumor associated antigen” (TAA) as used herein refers to aprotein which is present on tumor cells, and on normal cells duringfetal life (onco-fetal antigen), after birth in selected organs, or onmany normal cells, but at much lower concentration than on tumor cells.A variety of TAA have been described. An exemplary TAA is a mucin suchas MUC1, described in further detail below. In contrast, tumor specificantigen (TSA) (aka. “tumor-specific transplantation antigen” or TSTA)refers to a protein absent from normal cells. TSAs usually appear whenan infecting virus has caused the cell to become immortal and to expressa viral antigen(s). An exemplary viral TSA is the E6 or E7 proteins ofHPV type 16. TSAs not induced by viruses include idiotypes theimmunoglobulin idiotypes associated with B cell lymphomas or the T cellreceptor (TCR) on T cell lymphomas. TAAs are more common than TSAs.

Both TAA and TSA may be the immunological target of an expression vectorvaccine. Unless indicated otherwise, the term “tumor antigen” is usedherein to refer collectively to TAA and TSA.

The term “mucin” as used herein refers to any of a class of highmolecular weight glycoproteins with a high content of clusteredoligosaccharides O-glycosidically linked to tandem repeating peptidesequences which are rich in threonine, serine and proline. Mucin plays arole in cellular protection and, with many sugars exposed on theextended structure, effects multiple interactions with various celltypes including leukocytes and infectious agents. Mucin antigens alsoinclude those identified as CD227, Tumor-associated epithelial membraneantigen (EMA), Polymorphic epithelial mucin (PEM), Peanut-reactiveurinary mucin (PUM), episialin, Breast carcinoma-associated antigen DF3,H23 antigen, mucin 1, Episialin, Tumor-associated mucin,Carcinoma-associated mucin. Also included are CA15-3 antigen, M344antigen, Sialosyl Lewis Antigen (SLA), CA19-9, CA195 and other mucinantigen previously identified by monoclonal antibodies (e.g., see U.S.Pat. No. 5,849,876). The term mucin does not include proteoglycans whichare glycoproteins characterized by glycosaminoglycan chains covalentlyattached to the protein backbone.

At least 15 different mucins have been described including MU1, MUC2,MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC12, MUC13,MUC15, and MUC16 (these may also be designated with a hyphen between“MUC” and the number). The nucleotide sequence and amino acid sequenceof these mucins are known. The NCBI and Swiss Prot accession nos. foreach of these mucins are as follows: MUC1 (NCBI NM002456, Swiss ProtP15941), MUC2, (NCBI NM002457, Swiss Prot Q02817) MUC3A (NCBI AF113616,Swiss Prot Q02505), MUC3B (NCBI AJ291390, Swiss Prot Q9H195), MUC4 (NCBINM138299, Swiss Prot Q99102), MUC5AC (NCBI AF043909, Swiss Prot Q8WWQ5),MUC5B (Swiss Prot Q9HC84), MUC6 (NCBI U97698, Swiss Prot Q8N811), MUC7(NCBI L42983, Swiss Prot Q8TAX7), MUC8 (NCBI U14383, Swiss Prot Q12964),MUC9 (NCBI U09550, Swiss Prot Q12889), MUC12 (Swiss Prot Q9UKN1), MUC13(NCBI NM017648, Swiss Prot Q9H3R2), MUC15 (NCBI NM145650, Swiss ProtQ8WW41), and MUC16 (NCBI AF361486, Swiss Prot Q8WX17; aka CA125).

There are two structurally and functionally distinct classes of mucins:secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) andtransmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17). Theproducts of some MUC genes do not fit well into either class (MUC7,MUC8, MUC9, MUC13, MUC15, MUC16).

The characteristics of particular mucins as TAA in particular cancers issupported by alterations in expression and structure in association withpre-neoplastic and neoplastic lesions (Filipe M I: Invest Cell Pathol1979, 2:195-216; Filipe M I, Acta Med Port 1979, 1:351-365). Forinstance, normal mucosa of the stomach is characterized by theexpression of MUC1, MUC5A/C, MUC6 mRNA and the encoded immunoreactiveprotein. Also, high levels of MUC2, MUC3 mucin mRNA and encodedimmunoreactive protein are associated with intestinal metaplasia.Gastric cancer exhibits markedly altered secretory mucin mRNA levelscompared with adjacent normal mucosa, with decreased levels of MUC5 andMUC6 mRNA and increased levels of MUC3 and MUC4 mRNA. High levels ofMUC2 and MUC3 mRNA and protein are detectable in the small intestine,and MUC2 is the most abundant colonic mucin.

Mucins represent diagnostic markers for early detection of pancreaticcancer and other cell types. Studies have shown, that ductaladenocarcinomas (DACs) and tumor cell lines commonly overexpress MUCImucin. See Andrianifahanana et al., Clin Cancer Res 2001, 7:4033-4040).This mucin was detected only at low levels in the most chronicpancreatitis and normal pancreas tissues but is overexpressed in allstages of pancreatic cancers. The de novo expression of MUC4 inpancreatic adenocarcinoma and cell lines has been reported(Hollingsworth et al., Int J Cancer 1994, 57:198-203). MUC4 mRNAexpression has been observed in the majority of pancreaticadenocarcinoma and established pancreatic cancer cell lines but not innormal pancreas or chronic pancreatitis tissues. MUC4 expression alsohas been associated with lung cancer (see Nguyen et al. 1996 Tumor Biol.17:176-192). MUC5 is associated with metastases in non-small cell lungcancer (see Yu et al., 1996 Int. J. Cancer 69:457-465). MUC6 isoverexpressed and MUC5AC is de novo expressed in gastric and invasiveDACs (Kim et al., Gastroenterology 2002, 123:1052-1060). MUC7 has beenreported as a marker for invasive bladder cancer (see Retz et al. 1998Cancer Res. 58:5662-5666).

Expression of the MUC2 secreted gel-forming mucin is generally decreasedin colorectal adenocarcinoma, but preserved in mucinous carcinomas, adistinct subtype of colon cancer associated with microsatelliteinstability. MUC2 is increased in laryngeal cancer (Jeannon et al. 2001Otolaryngol Head Neck Surg. 124:199-202). Another secreted gel-formingmucin, MUC5AC, a product of normal gastric mucosa, is absent from normalcolon, but frequently present in colorectal adenomas and colon cancers.

MUC1, also known as episialin, polymorphic epithelial mucin (PEM), mucinlike cancer associated antigen (MCA), CA27.29, peanut-reactive urinarymucin (PUM), tumor-associated epithelial mucin, epithelial membraneantigen (EMA), human milk fat globule (HMFG) antigen, MUC1/REP,MUC1/SEC, MUC1/Y, CD227, is the most well known of the mucins. The geneencoding MUCI maps to 1q21-q24. The MUC1 gene contains seven exons andproduces several different alternatively spliced variants. The tandemrepeat domain is highly O-glycosylated and alterations in glycosylationhave been shown in epithelial cancer cells.

MUC1 mRNA is polymorphic in size. There are presently nine isoforms ofMUC1 based on alternate splicing (isoform no.: NCBI accession no.; 1: IDP15941-1, 2: ID P15941-2, 3: ID P15941-3, 4: ID P15941-4, 5: P15941-5,6: ID P15941-6, 7: ID P15941-7, 8: ID P15941-8, and 9: ID P15941-9).

MUC1 isoform 1 (aka. MUC1/REP) is a polymorphic, type I transmembraneprotein containing: 1) a large extracellular domain, primarilyconsisting of a 20-amino acid (aa) repeat motif (a region known asVariable Number (30-100) of tandem repeats—VNTR); 2) a transmembranedomain; and 3) a 72-aa cytoplasmic tail. During biosynthesis, theMUC1/REP protein is modified to a large extent, and a considerablenumber of O-linked sugar moieties confer mucin-like characteristics onthe mature protein. Soon after translation, MUC1/REP is cleaved into twoproducts that form a tightly associated heterodimer complex composed ofa large extracellular domain, linked noncovalently to a much smallerprotein including the cytoplasmic and transmembrane domains. Theextracellular domain can be shed from the cell. Using Swiss Prot P15941as a reference (see FIGS. 1A and 1B), the extracellular domain (ecm) ofMUC1 isoform 1 represents amino acids 24 to 1158, the transmembranedomain represents 1159-1181, and the cytoplasmic domain represents1182-1255. The SEA domain represents is 1034-1151 and represents aC-terminal portion of what is referred to as the extracellular domain.The SEA domain of a mucin is generally a target for proteolyticcleavage, yielding two subunits, the smaller of which is associated withthe cell membrane.

MUC1 isoform 5 (aka MUC1/SEC) is a form of MUC1 that is secreted bycells. It has an extracellular domain that is identical to that ofisoform 1 (MUC1/REP), but lacks a transmembrane domain for anchoring theprotein to a cell membrane. MUC1 isoform 7 (aka MUC1/Y) contains thecytoplasmic and transmembrane domains observed in isoforms 1 (MUC1/REP)and 5 (MUC1/SEC), but has an extracellular domain that is smaller thanMUC1, lacking the repeat motif and its flanking region (see Baruch A. etal., 1999 Cancer Res. 59, 1552-1561). Isoform 7 behaves as a receptorand binds the secreted isoform 5. Binding induces phosphorylation ofisoform 7 and alters cellular morphology and initiates cell signalingthrough second messenger proteins such as GRB2, (see Zrihan-Licht S. etal., 1995 FEBS Lett. 356, 130-136). It has been shown that β-catenininteracts with the cytoplasmic domain of MUC1 (Yamamoto M. et al., 1997J. Biol. Chem. 272, 12492-12494).

MUC1 is expressed focally at low levels on normal epithelial cellsurfaces. See 15. Greenlee, et al., Cancer Statistics CA Cancer J. 50,7-33 (2000); Ren, et al., J. Biol. Chem. 277, 17616-17622 (2002);Kontani, et al., Br. J. Cancer 84, 1258-1264 (2001); Rowse, et al.,Cancer Res. 58, 315 (1998). MUC1 is overexpressed in carcinomas of thebreast, ovary, pancreas as well as other carcinomas (see also Gendler S.J. et al, 1990 J. Biol. Chem. 265, 15286-15293). A correlation is foundbetween acquisition of additional copies of MUC1 gene and high mRNAlevels (p<0.0001), revealing the genetic mechanism responsible for MUC1gene overexpression, and supporting the role of MUC1 gene dosage in thepathogenesis of breast cancer (Biche I. et al.,. 1997 Cancer Genet.Cytogenet. 98, 75-80). MUC1 mucin, as detected immunologically, isincreased in expression in colon cancers, which correlates with a worseprognosis and in ovarian cancers.

High level expression of the MUC1 antigen plays a role in neoplasticepithelial mucosal cell development by disrupting the regulation ofanchorage dependent growth (disrupting E-cadherin function), which leadsto metastases. See Greenlee, et al., Cancer Statistics CA Cancer J. 50,7-33 (2000); Ren, et al. J. Biol. Chem. 277, 17616-17622 (2002).Non-MHC-restricted cytotoxic T cell responses to MUC1 have been reportedin patients with breast cancer. See Kontani et al., Br. J. Cancer 84,1258-1264 (2001). Human MUC1 transgenic mice (“MUC1.Tg”) have beenreported to be unresponsive to stimulation with human MUC1 antigen. SeeRowse, et al., Cancer Res. 58, 315 (1998). Human MUC1 transgenic miceare useful for evaluating the development of immunity to MUC1 as a selfantigen.

MUC1 protein and mRNA have been found in the ER-positive MCF-7 andBT-474 cells as well as in the ER-negative MDA-MB-231 and SK-BR-3 BCCcells. The mRNA Transcript level was higher in ER+ than in ER− celllines. MUC1 reacts with intracellular adhesion molecule-1 (ICAM-1). Atleast six tandem repeats of MUC1 are need (Regimbald et al., 1996 CancerRes. 56, 4244-4249). The tandem repeat peptide of MUC1 from T-47D BCCwas found to be highly O-glycosylated with 4.8 glycosylated sites perrepeat, which compares to 2.6 sites per repeat for the mucin from milk.

The term “mucin antigen” as used herein refers to the full length mucinall or a portion of the mucin that contains an epitope characterized inbeing able to elicit cellular immunity using a MUC-CD40L expressionvector administered in vivo as described herein. A “mucin antigen”includes one or more epitopes from the extracellular domain of a mucinsuch as one or more of the tandem repeat motifs associated with theVNTR, or the SEA region. A mucin antigen may contain the entireextracellular domain. Also included within the meaning of “mucinantigen” are variations in the sequence including conservative aminoacid changes and the like which do not alter the ability of the antigento elicit an immune response that crossreacts with a native mucinsequence.

The VNTR consists of variable numbers of a tandemly repeated peptidesequences, which differ in length (and composition) according to agenetic polymorphism and the nature of the mucin. The VNTR may alsoinclude 5′ and 3′ regions, which contain degenerate tandem repeats. Forexample, in MUC1, the number of repeats varies from 21 to 125 in thenorthern European population. In the U.S. the most infrequent allelescontains 41 and 85 repeats, while more common alleles have 60-84repeats. The MUC1 repeat has the general repeating peptide sequencePDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 3). Underlying the MUC1 tandem repeatis a genetic sequence polymorphism at three positions shown bolded andunderlined (positions 2, 3 and 13). The concerted replacement DT→ES(sequence variation 1) and the single replacements P→Q (sequencevariation 2), P→A (sequence variation 3), and P→T (sequence variation 4)have been identified and vary with position in the domain (see Engelmannet al., 2001 J. Biol. Chem. 276:27764-27769). The most frequentreplacement DT.fwdarw.ES occurs in up to 50% of the repeats. Table 1shows some exemplary tandem repeat sequences.

TABLE 1 Mucin Tandem Repeat Sequences Mucin Tandem Repeat (SEQ ID NO:)Mucin source MUC1 PDTRPAPGSTAPPAHGVTSA Mammary (SEQ ID NO: 3)PDNKPAPGSTAPPAHGVTSA Pancreatic (SEQ ID NO: 33) MUC2PTTTPPITTTTTVTPTPTPTGTQT Intestinal (SEQ ID NO: 4) Tracheobronchial MUC3HSTPSFTSSITTTETTS (SEQ ID NO: 5) Intestinal Gall Bladder MUC4TSSASTGHATPLPVTD (SEQ ID NO: 6) Colon Tracheobronchial MUC5AC TTSTTSAP(SEQ ID NO: 7) Gastric Tracheobronchial MUC5B SSTPGTAHTLTMLTTTATTPTATGTracheobronchial STATP (SEQ ID NO: 8) Salivary MUC7TTAAPPTPSATTPAPPSSSAPG Salivary (SEQ ID NO: 9) MUC8TSCPRPLQEGTPGSRAAHALSRRGHR Tracheobronchial VHELPTSSPGGDTGF (SEQ ID NO:10)

Although a mucin antigen as used herein may comprise only a singletandem repeat sequence motif, it should be understood that the immuneresponse will generally be stronger and more efficiently generated ifthe vector encodes multiple such repeats. The invention vectorpreferably encodes mucin tandem repeats from 2-4, more preferably from5-9, even more preferably from 10-19, yet even more preferably from20-29, still more preferably from 30-39, and still yet more preferablyfrom 40-50. Tandem repeats greater than 50 are possible and may includethe number of such repeats found in natural mucins.

A mucin antigen as this term is used herein also may encompass tandemrepeats from different types of mucins. For example, an expressionvector may encode tandem repeats from two different mucins, e.g., MUC1and MUC2. Such a vector also may encode multiple forms of the SEA domainas well or a combination of tandem repeats and one or more SEA domains.

A secretable form of a mucin is one which lacks all or substantially allof its transmembrane domain. The transmembrane domain of a mucin, ifpresent, is generally about 24 amino acids in length and functions toanchor the mucin or a fragment of the mucin in the cell membrane. Asecretable form of MUC1 in which all of the transmembrane domain hasbeen deleted is MUC1 missing residues 1159-1181. A mucin missingsubstantially all of the transmembrane is one where the domain comprises6 residues or less of sequence at one end of the transmembrane domain,more preferably less than about 4 residues of sequence at one end of thetransmembrane domain, even more preferably less than about 2 residues ofsequence on one end of the transmembrane domain, and most preferably 1residue or less on one end of the transmembrane domain. Thus, a mucinthat lacks substantially all of the transmembrane domain rendering themucin secretable is one that contains no more than six residues ofsequence on one end of the transmembrane domain. In a preferredembodiment, the vaccine vector transcription unit encodes a secretableform of mucin lacking the entire transmembrane domain.

It should be understood that a mucin which lacks a functionaltransmembrane domain may still include all or a portion of thecytoplasmic domain and all or a portion of the SEA region, if present.

A source of DNA encoding the various mucins, and mucin antigens may beobtained from mucin expressing cell lines using a commercial cDNAsynthesis kit and amplification using a suitable pair of PCR primersthat can be designed from the published mucin DNA sequences. Forexample, MUC1 or MUC2 encoding nucleic acid may be obtained fromCRL-1500 cells, available from the American Type Culture Collection.Mucin encoding DNA also may be obtained by amplification from RNA orcDNA obtained or prepared from human or other animal tissues. For DNAsegments that are not that large, the DNA may be synthesized using anautomated oligonucleotide synthesizer.

The term “linker” as used herein with respect to the transcription unitof the expression vector refers to one or more amino acid residuesbetween the mucin antigen and CD40 ligand. The composition and length ofthe linker may be determined in accordance with methods well known inthe art and may be tested for efficacy. See e.g. Arai et al., “Design ofthe linkers which effectively separate domains of a bifunctional fusionprotein” Protein Engineering, Vol. 14, No. 8, 529-532, August 2001. Thelinker is generally from about 3 to about 15 amino acids long, morepreferably about 5 to about 10 amino acids long, however, longer orshorter linkers may be used or the linker may be dispensed withentirely. Longer linkers may be up to about 50 amino acids, or up toabout 100 amino acids. A short linker of less than 10 residues ispreferred when the mucin antigen is N-terminal to the CD40 ligand.

The term “CD40 ligand” (CD40L) as used herein refers a full length orportion of the molecule known also as CD154 or TNF5. CD40L is a type IImembrane polypeptide having a cytoplasmic domain at its N-terminus, atransmembrane region and then an extracellular domain at its C-terminus.Unless otherwise indicated, the full length CD40L is designated hereinas “CD40L,” “wt CD40L” or “wtTmCD40L.” The form of CD40L in which thecytoplasmic domain has been deleted is designated herein as ΔCtCD40L.”The form of CD40L where the transmembrane domain has been deleted isdesignated herein as ΔTmCD40L.” The form of CD40L where both thecytoplasmic and transmembrane domains have been deleted is designatedherein as ΔCtΔTmCD40L.” The nucleotide and amino acid sequence of CD40Lfrom mouse and human is well known in the art and can be found, forexample, in U.S. Pat. No. 5,962,406 (Armitage et al.). Also includedwithin the meaning of “CD40 ligand” are variations in the sequenceincluding conservative amino acid changes and the like which do notalter the ability of the ligand to elicit an immune response to a mucinin conjunction the fusion protein of the invention.

Murine CD40L (mCD40L) is 260 amino acids in length. The cytoplasmic (Ct)domain of mCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-260.

Human CD40L (hCD40L) is 261 amino acids in length. The cytoplasmicdomain of hCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-261.

A secretable form of CD40 ligand is one which is missing all orsubstantially all of its transmembrane domain. The transmembrane domainof CD40L, which contains about 24 amino acids in length, functions toanchor CD40 ligand in the cell membrane. CD40L from which all of thetransmembrane domain has been deleted is CD40 ligand lacking residues23-46. CD40 ligand missing substantially all of the transmembrane is onethat retains 6 residues or less of sequence at one end of thetransmembrane domain, more preferably less than about 4 residues ofsequence at one end of the transmembrane domain, even more preferablyless than about 2 residues of sequence on one end of the transmembranedomain, and most preferably 1 residue or less on one end of thetransmembrane domain. Thus, a CD40L that lacks substantially all of thetransmembrane domain rendering the CD40L secretable is one that retainsno more than six residues of sequence on one end of the domain. Such asCD40L would contain, in addition to the extracellular domain andoptionally the cytoplasmic domain, and no more than amino acids 41-46 or23-28 located in the transmembrane domain of CD40L. In a preferredembodiment, the vaccine vector transcription unit encodes a secretableform of CD40 containing less than 10% of the transmembrane domain. Morepreferably, CD40L contains no transmembrane domain.

It should be understood that a CD40L which lacks a functionaltransmembrane domain may still include all or a portion of thecytoplasmic domain Likewise, a CD40L which lacks a functionaltransmembrane domain may include all or a substantial portion of theextracellular domain.

As used herein, an expression vector of the present invention can beadministered as a vaccine to induce immunity to a mucin. The vector maybe formulated as appropriate with a suitable pharmaceutically acceptablecarrier. Accordingly, the vectors may be used in the manufacture of amedicament or pharmaceutical composition. Expression vectors may beformulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use.Liquid formulations may be buffered, isotonic, aqueous solutions.Powders also may be sprayed in dry form. Examples of suitable diluentsare normal isotonic saline solution, standard 5% dextrose in water, orbuffered sodium or ammonium acetate solution. Such formulations areespecially suitable for parenteral administration, but may also be usedfor oral administration or contained in a metered dose inhaler ornebulizer for insufflation. It may be desirable to add excipients suchas polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,polyethylene glycol, mannitol, sodium chloride, sodium citrate, and thelike.

Alternately, vectors may be prepared for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe vectors. Solid carriers include starch, lactose, calcium sulfatedihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin,acacia, agar or gelatin. Liquid carriers include syrup, peanut oil,olive oil, saline and water. The carrier may also include a sustainedrelease material such as glyceryl mono stearate or glyceryl distearate,alone or with a wax. The amount of solid carrier varies but, preferably,will be between about 20 mg to about 1 g per dosage unit. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, or an aqueous or non-aqueous suspension.

Vectors of the invention may be formulated to include other medicallyuseful drugs or biological agents. The vectors also may be administeredin conjunction with the administration of other drugs or biologicalagents useful for the disease or condition that the invention compoundsare directed.

As employed herein, the phrase “an effective amount,” refers to a dosesufficient to provide concentrations high enough to generate (orcontribute to the generation of) an immune response in the recipientthereof. The specific effective dose level for any particular subjectwill depend upon a variety of factors including the disorder beingtreated, the severity of the disorder, the activity of the specificcompound, the route of administration, the rate of clearance of theviral vectors, the duration of treatment, the drugs used in combinationor coincident with the viral vectors, the age, body weight, sex, diet,and general health of the subject, and like factors well known in themedical arts and sciences. Various general considerations taken intoaccount in determining the “therapeutically effective amount” are knownto those of skill in the art and are described, e.g., in Gilman et al.,eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1990. For administration ofvectors, the range of particles per administration typically if fromabout 1×10⁷ to 1×10¹¹, more preferably 1×10⁸ to 5×10¹⁰, and even morepreferably 5×10⁸ to 2×10¹⁰. A vector can be administered parenterally,such as intravascularly, intravenously, intraarterially,intramuscularly, subcutaneously, or the like. Administration can also beorally, nasally, rectally, transdermally or inhalationally via anaerosol. The vectors may be administered as a bolus, or slowly infused.Vector are preferably administered subcutaneously.

The invention recombinant expression vectors offer a potentiallysuperior approach that allows a higher efficiency of gene transfer thanthat of DNA vaccines. As demonstrated herein, adenoviral vectorsencoding tumor associated antigens can induce a protective cellular andhumoral immunity against such antigens, including those to whichtolerance had developed. Although not wishing to be bound by any theory,it is believed that the invention vaccines facilitated DCs maturation,promoting the development of effective antigen-specific immunity. It isalso demonstrated herein that the secretable fusion protein encoding theextracellular domain of human MUC1 and the murine CD40L lacking atransmembrane and cytoplasmic domain (i.e. ecdhMUC1-ΔCtΔTmCD40L)produced from an adenoviral vector dramatically enhanced the potency ofthe cellular immune response to MUC1 expressing tumor cells. Althoughnot wishing to be bound by any theory, it is believed that subcutaneousinjection of the Ad-K-ecdhMUC1-ΔCtΔTmCD40L vector elicited strong MUC1specific CD8⁺ T cell-mediated immunity, which prevented the engraftmentof cancer cells that expressed the MUC1 tumor associated antigen.

The immunity generated against the mucin antigen using the inventionvector vaccine is long lasting. As used herein, the term “long lasting”means that immunity elicited to the mucin antigen encoded by the vectorcan be demonstrated for up to 6 months from the last administration,more preferably for up to 8 months, more preferably for up to one year,more preferably up to 1.5 years, and more preferably for at least twoyears.

In one embodiment, immunity to a mucin TAA can be generated by producinga fusion protein that comprises the extracellular domain of MUC1 fusedto the amino-terminal end of the CD40 ligand from which thetransmembrane and cytoplasmic domains were deleted. Construction of suchvector is disclosed in the Examples. As was observed herein,subcutaneous administration of this adenoviral vector mucin vaccineinduced a very robust and long lasting CD8⁺ cytotoxic T cell lymphocytedependent systemic immune response against cancer cells which carry theMUC1 antigen. The mucin vaccine induced the production of memory cells,which underlie the long lasting immunity.

It was observed that vaccination of mice with the adenoviral vectorAd-sig-ecdhMUC1/ecdmCD40L induced an immune response which suppressedthe growth of human MUC1 (hMUC1) antigen positive tumor cells in 100% ofmice transgenic for hMUC1 (i.e. these mice are anergic to the hMUC1antigen prior to the vector injection. See Rowse, et al., Cancer Res.58, 315 (1998). These results demonstrated that theAd-sig-ecdhMUC1-ecd/ecdCD40L vector can be used for treating epithelialmalignancies that express the MUC1.

Subcutaneous injection of the adenoviral MUC1 expression vectorincreased the level of hMUC1 specific T cells in the spleens of injectedhMUC1 transgenic mice by 250 fold. The transgenic mice were anergic tothe hMUC1 antigen prior to the vector injection. Thus, vector injectionovercame the anergy, inducing a CD8⁺ T cell dependent systemic Th1immune response that was antigen specific, and HLA restricted. Theability to overcome anergy as observed for vaccination with theadenoviral MUC1 expression vector was not observed when transgenic micewere vaccinated with purified ecdhMUC1/ecdCD40L-HIS protein.

Although not wishing to be bound by any theory, it is believed that thecells infected in the vicinity of the site of subcutaneous injection ofthe vector release the mucin antigen/CD40 ligand secretory which istaken up by antigen presenting cells (e.g. DCs) in the vicinity of theinfected cells. The internalized mucin antigen would be digested in theproteosome with the resultant mucin antigen peptides trafficking to theendoplasmic reticulum where they would bind to Class I MHC molecules.Eventually, the DCs would present the mucin antigen on the surface inthe Class I MHC molecule. Activated, tumor antigen-loaded antigenpresenting cells would migrate to lymphocyte bearing secondary organssuch as the regional lymph nodes or the spleen. During the two weeks ofcontinuous release of the mucin antigen/CD40 ligand protein, CD8cytotoxic T cell lymphocytes competent to recognize and kill cells whichcarried the tumor associated antigens would be expanded in the lymphnodes and spleen by the presence of the activated and antigen loadeddendritic cells. The continuous nature of the stimulation and theexpansion of the mucin antigen specific cytotoxic T cells by thecontinuous release from the vector infected cells is believed togenerate an immune response which would be greater in magnitude than ispossible using a vector which carried a mucin antigen/CD40 ligand whichis non-secretory.

The methods of the present invention, therefore, can be used to generateimmunity to mucin which is a self-antigen in an individual. For example,a vector of the invention that encodes a mucin antigen from MUC1 can beused to generate CD8⁺ immunity in a human where the MUC1 mucin antigenis a self antigen. The invention methods also can be used to overcome astate of immunological anergy to a mucin which is a self-antigen.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES 1. Construction of Adenoviral Expression Vectors

The transcription unit, sig-ecdhMUC1-ΔCtΔTmCD40L of the adenoviralvector encodes a signal sequence (from an Ig kappa chain) followed bythe extracellular domain of human MUC1 which is connected via a linkerto a fragment of the CD40 ligand (human or mouse) which contains theextracellular domain without the transmembrane or cytoplasmic domains.The fusion protein was engineered to be secreted from vector infectedcells by the addition of the kappa chain signal sequence to theamino-terminal end of the fusion protein.

The amino acid sequence of human MUC-1 and the encoding nucleotidesequence are shown in FIGS. 2 and 1A and 1B, respectively. The encodedMUC1 protein represents 1255 amino acids encoded by nucleotides 74 to3,841 of SEQ ID NO: 1. The first 23 amino acids (encoded by 74 to 142 ofSEQ ID NO:1) represent the MUC1 signal sequence which is removed fromthe mature mucin. The extracellular domain represents about 1135 aminoacids from positions 24 to 1158 (encoded by nucleotides 143 to 3547).The tandem repeat region represents approximately 900 amino acids. Aminoacids 74 to 126 (encoded by 229 to 451 of SEQ ID NO:1) represents a 5′degenerate tandem repeat region, amino acids 127 to 945 represents thetandem repeat region (encoded by 452 to 2,908 of SEQ ID NO: 1) whileamino acids 946 to 962 represent a 3′ degenerate tandem repeat region(encoded by 2809 to 2959 of SEQ ID NO:1). The SEA domain representsamino acids 1034 to 1151, the transmembrane domain represents 1159 to1181, and the cytoplasmic domain represents 1182 to 1255 (see SEQ IDNO:2).

The transcription unit was introduced into the E1 gene region of theadenoviral vector backbone. After the adenoviral vector particles weregenerated in HEK 293 cells, the vector DNA was purified by cesiumchloride gradient centrifugation. The presence of the signal peptide inthe adenoviral vector was confirmed by restriction enzyme analysis andby DNA sequencing.

A transcription unit that included DNA encoding the signal sequence ofthe mouse IgG kappa chain gene upstream of DNA encoding human MUC-1(“sig-ecdhMUC-1”) was generated by PCR using plasmid pcDNA3-hMUC-1 (giftof Finn O. J., University of Pittsburgh School of Medicine) and thefollowing primers: DNA encoding the mouse IgG kappa chainMETDTLLLWVLLLWVPGSTGD (single letter amino acid code) (SEQ ID NO: 11)was prepared by PCR amplification (SEQ ID NOs: 12, 13 and 14) togenerate the full 21 amino acid mouse IgG kappa chain signal sequence(the start codon “ATG” is shown bolded in SEQ ID NO:12).

(SEQ ID NO: 12) 5′-CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTACTG CTG-3′ (SEQ ID NO: 13)5′-TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3′ (SEQ ID NO: 15)5′-TG CTC TGG GTT CCA GGT TCC ACT GGT GAG GAT G-3′ (SEQ ID NO: 15)5′-GGT TCC ACT GGT GAC GAT GTC ACC TCG GTC CCA GTC-3′(forward primer for MUC-1 repeat region) (SEQ ID NO: 16)5′-GAGCTCGAG ATT GTG GAC TGG AGG GGC GGT G-3′(reverse primer for MUC-1 repeat region)

sig-ecdhMUC-1 with the upstream kappa signal sequence was generated byfour rounds of PCR amplification (1^(st) round: primers SEQ ID NOs 15and 16, 2^(nd) round: primer SEQ ID NOs 14 and 16; 3^(rd) round: primerSEQ ID NOs 13 and 16; 4^(th) round: primer SEQ ID NOs 12 and 16). Thesig-ecdhMUC-1 encoding DNA was cloned into the pcDNA™ 3.1 TOPO vector(Invitrogen, San Diego, Calif.) forming pcDNA-sig-ecdhMUC-1.

pShuttle-ΔCtΔTmCD40L (no signal sequence and murine CD40L) was preparedas follows: Plasmid pDC406-mCD40L was purchased from the American TypeCulture Collection. A pair of PCR primers (SEQ ID NOs: 17 and 18) wasdesigned to amplify the mouse CD40 ligand from position 52 to 260 (i.e.,without the cytoplasmic and transmembrane domains) and include sequenceencoding a linker (indicated as “+ spacer”) at the 5′ end of theamplicon.

Mouse ΔCtΔTmCD40L+ spacer forward primer (MCD40LSPF) (CD40L sequenceitalicized; cloning site underlined and bolded):

(SEQ ID NO: 17) 5′-CCGCTCGAGAACGACGCACAAGCACCAAAATCAAAGGTCGAAGAGGAAGTA-3′Mouse CD40L reverse primer (MCD40LR; cloning site underlined)

(SEQ ID NO: 18) 5′-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAGTAA GCC AAAAGA TGA G-3′

The forward primer MCD40LSPF encodes a 10 residue spacer (LENDAQAPKS;single letter code; SEQ ID NO: 19) to be located between the mucin andthe CD40 ligand (mCD40L) of the transcription unit. PCR performed usingthe forward and reverse primers (SEQ ID NOs 17 and 18) and plasmidpDC406-mCD40L as the template resulted in PCR fragment“space+ΔCtΔTMCD40L”, which was inserted into the plasmidpcDNA-sig-ecdhMUC1 after restriction endonuclease digestion with XbaI(TCTAGA) and Xho I (CTCGAG). This vector is designatedpcDNA-sig-ecdhMUC1/ΔCtΔTmCD40L. A vector was produced that was otherwisethe same except that it encoded full length CD40L rather than thetruncated form. This vector was made using a CD40 forward primer thatannealed to the starting codons of murine CD40L. This vector isdesignated pShuttleCD40L (no signal sequence).

The sig-ecdhMUC1/ΔCtΔTmCD40L encoding DNA was cut from the pcDNA3TOPOvector using HindIII-XbaI restriction and inserted into pShuttle-CMV(see Murphy et al., Prostate 38: 73-78, 1999) downstream of the CMVpromoter. The plasmid is designated pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40L.Thus, the transcription unit sig-ecdhMUC1-ΔCtΔTmCD40L encodes the mouseIgG kappa chain secretory signal followed by the extracellular domain ofhuman MUC1 followed by a 10 amino acid linker with (NDAQAPK; SEQ ID NO:19) followed by murine CD40 ligand residues 52-260.

In some vectors, the mouse HSF1 trimer domain was added between theecdhMUC1 encoding DNA and ΔCtΔTm CD40L by PCR using plasmidpcDNA-sig-ecdhMUC1/ΔCtΔTmCD40L and the following primers:

(SEQ ID NO: 20) 5′-AAC AAG CTC ATT CAG TTC CTG ATC TCA CTG GTGGGATCC AAC GAC GCA CAA GCA CCA AAA TC-3′ (SEQ ID NO: 21)5′-AGC CTT CGG CAG AAG CAT GCC CAG CAA CAG AAAGTC GTC AAC AAG CTC ATT CAG TTC CTG-3′ (SEQ ID NO: 22)5′AAT GAG GCT CTG TGG CGG GAG GTG GCC AGC CTT CGG CAG AAG CAT G-3′(SEQ ID NO: 23) 5′GAT ATC CTC AGG CTC GAG AAC GAC GCA CAA GCACCA AAA GAG AAT GAG GCT CTG TGG CGG G-3′ (SEQ ID NO: 18)5′-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAG TAA GCC AAA AGA TGA G-3′.

HSF1/ΔCtΔTm CD40L with the trimer domain sequence was generated by fourrounds of PCR amplification (1^(st) round: primers SEQ ID NOs 23 and 18;2^(nd) round: primer SEQ ID NOs 22 and 18; 3^(rd) round: primer SEQ IDNOs 21 and 18; 4^(th) round: primer SEQ ID NOs 20 and 18). TheHSF1/ΔCtΔTm CD40L encoding DNA was cloned into pcDNA-sig-hMUC-1restriction sites XbaI (TCTAGA) and Xho I (CTCGAG). The sequence betweenMUC1 and mCD40L is as follows:

LENDAQAPKENEALWREVASFRQKHAQQQKVVNK LIQFLISLVGSNDAQAPKS (SEQ ID NO: 24),wherein the underlined segment is the trimer sequence which is bonded bythe linker LENDAQAPK (SEQ ID NO:25) and NDAQAPKS (SEQ ID NO:26).

In some vectors, a His tag encoding sequence was added to the end of theΔCtΔTm CD40L and was generated by PCR using Plasmid pDC406-mCD40L(purchased from the American Type Culture Collection) and the followingprimers:

Vector/ΔCtΔTm CD40L/His with the His tag sequence was generated by 2rounds of PCR amplification (1^(st) round: primers 1+2; 2^(nd) round:primer 1+3). The /ΔCtΔTmCD40L/His encoding DNA was cloned intopcDNA-sig-ecdhMUC-1 restriction sites XbaI (TCTAGA) and Xho I (CTCGAG).

The recombinant adenoviral vectors were generated using the AdEasyvector system (Stratagene, San Diego, Calif.). Briefly the resultingplasmid pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40L, and other control adenoviralvectors were linearized with Pme I and co-transformed into E. colistrain BJ5183 together with pAdEasy-1, the viral DNA plasmid.Recombinants were selected with kanamycin and screened by restrictionenzyme analysis. The recombinant adenoviral construct was then cleavedwith Pac I to expose its Inverted Terminal Repeats (ITR) and transfectedinto 293A cells to produce viral particles. The titer of recombinantadenovirus was determined by the Tissue culture Infectious Dose (TCID₅₀)method.

Primers for amplifying human ΔCtΔTmCD40L+ spacer using a human CD40ligand cDNA template are set forth below.

Human ΔCtΔTmCD40L+ spacer forward primer (HCD40LSPF) (CD40L sequenceitalicized):

Human CD40L reverse primer (HCD40LR)

These primers will amplify a ΔCtΔTmCD40L+ spacer which encodes 47-261 ofhuman CD40L. The forward primer HCD40LSPF encodes a 10 residue spacer(LENDAQAPKS; single letter code; SEQ ID NO: 19) to be located betweenthe tumor antigen and the CD40 ligand (hCD40L) of the transcriptionunit. PCR performed using the forward and reverse primers (SEQ ID NOs 30and 31) and Plasmid pDC406-hCD40L as the template results in PCRfragment “space+ΔCtΔTmCD40L(human),” which is inserted into the plasmidpcDNA-sig-ecdhMUC1 after restriction endonuclease digestion with XbaI(TCTAGA) and Xho I (CTCGAG). The sig-ecdhMUC1/ΔCtΔTmCD40L (human)encoding DNA was cut from the pcDNA3TOPO using HindIII-XbaI restrictionand inserted into pShuttle-CMV (see Murphy et al., Prostate 38: 73-78,1999) downstream of the CMV promoter. This vector is designated pShuttlesig-ecdhMUC1/ΔCtΔTmCD40L(human). Modification of pShuttlesig-ecdhMUC1/ΔCtΔTmCD40L(human) to include the ecdhMUC1 upstream of thehuman CD40 ligand sequence was accomplished essentially as describedabove for the murine CD40 ligand encoding vectors. Thus, thetranscription unit sig-ecdhMUC1-ΔCtΔTmCD40L(human) encodes the kappasecretory signal followed by the extracellular domain of human MUC1followed by a 10 amino acid linker (NDAQAPK; SEQ ID NO:19) followed byhuman CD40 ligand residues 47-261.

In an alternative approach, DNA encoding the human growth hormone signalsequence MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQID NO: 32) could be used in place of the kappa chain signal sequence.

2. Overcoming Anergy to MUC1 in MUC1 Transgenic Mice

a) Cytokine Production of Adenoviral Infected DCs

Bone marrow derived DCs was harvested from hMUC-.Tg transgenic mice at48 hours after exposure to the adenoviral vectors. The cells wereexposed to vector at MOI 100, and plated in 24-well plates at 2×10⁵cells/ml. After incubation for 24 hours at 37° C., supernatant fluid (1ml) was harvested and centrifuged to remove debris. The level of murineIL-12 or IFN-gamma released into the culture medium was assessed byenzyme-linked immunoadsorbent assay (ELISA) using the mouse IL-12 p70 orIFN-gamma R & D Systems kits.

Bone marrow derived DCs contacted with the Ad-sig-ecdmMUC1-ΔCtATCD40L(murine) vector showed significantly increased the levels of interferongamma and IL-12 cytokines from DCs harvested from the hMUC-.Tgtransgenic mice at 48 hours after exposure to the vector. In contrast,virtually no cytokines were detected from restimulated DC's from animalsimmunized with an adenoviral vector that encoded the extracellulardomain of hMUC1 but without fusion to a secretable form of CD40L. Theseresults indicate that the ecdhMUC1/ecdmCD40L (murine) fusion proteinforms functional trimers and binds to the CD40 receptor on DCs.

b) Evaluation of Trimer Formation by ecdhMUC1-HSF1-ΔCtΔTmCD40L FusionProtein Expressed from Ad-sig-ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS

Trimerization of ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS fusion protein wasevaluated following release from cells transformed withAd-sig-ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS vector. The expressed fusionprotein was purified from the supernatant of 293 cells exposed to thevector using a His Tag purification kit. Nondenaturing gelelectrophoresis showed a molecular weight consistent with trimer.formation.

c) Effect of Ad-sig-ecdhMUC1-ΔCtΔTmCD40L Vector Injection onEstablishment of MUC1 Expressing Cancer Cells

hMUC1.Tg mice injected subcutaneously with theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector were resistant toengraftment by the hMUC1 positive LL2/LL1hMUC1 mouse cancer cells.Control animals not injected with vector were not resistant to thegrowth of the same cells. Also, hMUC1.Tg mice injected with theAd-sig-ecdhMUC1/ecdCD40L (murine) vector were not resistant toengraftment by parental cell line (LL2/LL1), which does not expressMUC1.

hMUC1.Tg mice injected intravenously with ecdhMUC1-ΔCtΔTmCD40L (murine)protein were not resistant to engraftment by the hMUC1 positiveLL2/LL1hMUC1 mouse cancer cells. Furthermore, hMUC1.Tg mice injectedwith Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector lived longer than didcontrol vector injected mice subsequently administered the LL2/LL1hMUC1cell line.

3. Cellular Mechanisms Underlying Breakdown of Anergy

a) Cytokine Release from Vaccinated vs. Non Vaccinated Mice

A population of splenic CD8⁺ T lymphocytes was obtained seven daysfollowing Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector administration wasobtained by depleting CD4⁺ T lymphocytes using CD4⁺ antibody coatedmagnetic beads. The isolated CD8⁺ T lymphocytes released over 2,000times the level of interferon gamma as did CD8⁺ T cells from MUC1.Tgmice administered a control vector (without MUC1).

b) Cytotoxicity Assay

Splenic T cells collected from hMUC1.Tg mice 7 days followingadministration of Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector werecultured with hMUC1 antigen positive LL2/LL1hMUC1 cancer cells in vitrofor 7 days. The stimulated splenic T cells were mixed in varying ratioswith either the hMUC1 positive LL2/LL1hMUC1 cells or the hMUC1 negativeLL2/LL1 cancer cells. The results showed that T cells fromAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector vaccinated mice werecytotoxic only for the cancer cells expressing hMUC1.

c) Ad-sig-ecdhMUC1-ΔCtΔTmCD40L Vector Injection Overcomes Resistance toExpansion of hMUC1 Specific T Cells

DCs obtained in vitro from bone marrow cells were exposed to theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector for 48 hours. Splenic CD8⁺ Tcells, obtained from hMUC1.Tg transgenic mice 7 days following no vectorinjection or subcutaneous injection with the Ad-sig-ecdhMUC1-ΔCtΔTmCD40L(murine) vector, were mixed in a 1/1 ratio with theAd-sig-ecdhMUC1/ecdCD40L (murine) vector-infected DCs. The ERK1/EK2proteins, the endpoint of the Ras/MAPK signaling pathway, werephosphorylated in the CD8+ T cells isolated fromAd-sig-ecdhMUC1-ΔCtΔTmCD40L vector injected hMUC1.Tg transgenic micefollowing 45 minutes of in vitro exposure to Ad-sig-ecdhMUC1-ΔCtΔTmCD40L(murine) vector infected DCs. In contrast no increase in phosphorylationof ERK1 and ERK2 proteins was seen in CD8 positive T cells fromunvaccinated hMUC1.Tg mice. These results demonstrate that CD8 positiveT cells from MUC1.Tg transgenic mice vaccinated with theAd-sig-ecdMUC1-ΔCtΔTmCD40L (murine) vector were no longer anergic toMUC1.

4. Tumor Immunotherapy by Vaccination with Vector Encoding a MUC1-CD40LFusion Protein

For a tumor prevention protocol, animals were administeredAd-sig-ecdhMUC-1/ecdCD40L vector on day 1, 7 and 21. Three weeks later,the animals were administered LL2/LL1hMUC-1 tumor cells subcutaneously.Two weeks later, mice were administered intravenously 500,000LL2/LL1hMUC-1 tumor cells via the tail vein. The size of thesubcutaneous tumor nodules which developed, were measured by caliper atmultiple time points to determine the effect of the various vaccineschedules on the growth of the LL2/LL1hMUC-1 cells as subcutaneousnodules. Lung metastases were measured by lung total weight followingsacrifice.

Subcutaneous tumor measurements were made at various time points. Vectorvaccinated mice completely suppressed the appearance of subcutaneousLL2/LL1hMUC-1 tumor. Vector vaccinated animals also effectivelysuppressed the growth of metastatic cancer nodules developing in thelungs.

A tumor treatment (post establishment) protocol was evaluated. In thisschedule, subcutaneous tumor (500,000 of the LL2/LL1hMUC-1) wasadministered on day 1 and vaccinations were carried out at day 5. Vectorwas administered on days 5, 12 and 26. Tumor was administered i.v. onday 35 and tumor development (subcutaneous and lung) evaluated at day49. Reduction in the size of the subcutaneous tumor and the extent oflung metastatic nodules was reduced in vector vaccinated animals.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A plasmid expression vector for generating in anindividual immunity against one or more mucin antigens, said vectorcomprising a transcription unit, said transcription unit comprising anucleic acid operably linked to a promoter, said transcription unitencoding a secretable polypeptide, said encoded polypeptide comprisingfrom the amino terminus, a secretory signal sequence, one or more mucinantigen(s) or fragments thereof, and a CD40 ligand, wherein the one ormore mucin antigen(s) or fragments thereof are connected together,wherein (i) the one or more mucin antigen(s) or fragments thereof andthe CD40 ligand lack all or substantially all of their transmembranedomains; (ii) one end of the connected, one or more mucin antigen(s) orfragments thereof is attached to the amino terminus of the extracellulardomain of said CD40 ligand; and, (iii) said encoded polypeptide isconfigured to promote presentation on Class I MHC and activation withexpansion of mucin-specific, Class I MHC restricted, cytotoxic Tlymphocytes.
 2. A plasmid expression vector according to claim 1,wherein a liposome is used as a delivery vehicle for said expressionvector.
 3. An expression vector for generating in an individual immunityagainst a mucin antigen, said vector comprising a transcription unitencoding a secretable polypeptide, said encoded polypeptide comprisingfrom the amino terminus, a secretory signal sequence, the mucin antigen,and CD40 ligand, wherein (i) the mucin antigen is missing all orsubstantially all of the transmembrane domain; (ii) the CD40 ligand ismissing all or substantially all of the transmembrane domain; (iii) themucin antigen is attached to the amino-terminus of the extracellulardomain of said CD40 ligand; and (iv) wherein said encoded polypeptide isconfigured to promote presentation of Class I MHC and activation withexpansion of mucin-specific, Class I MHC restricted, cytotoxiclymphocytes, and wherein said expression vector is present in a liposomefor delivering said expression vector.